In the coming year we plan to explore the evolutionary origins of the human and other mammalian carbonic anhydrase isozymes (CA I, CA II, and CA III) by not only continuing our sequence analysis of selected mammalian CA isozymes, but also by determining the sequences of avian and reptilian CA isozymes. Our work on the recently discovered CA isozyme from mammalian muscle, CA III, will be directed toward studies on its active site mechanisms with the hope of learning more about the natural physiological substrate of this muscle specific isozyme of carbonic anhydrase. A number of recently discovered inherited electrophoretic variants of human red cell CA I and CA II will be studied in order to determine the amino acid substitutions in these mutant CA isozymes as well as to chemically characterize their activities and stabilities. Work on carbonic anhydrase mRNA will involve the preparation of labelled cDNA probes which will be used in studies on both the regulation of the CA I and CA II isozymes, but also the organization (mapping) of the two closely linked genes.